CN115436520B - Construction and detection method of luffa UPLC characteristic spectrum - Google Patents
Construction and detection method of luffa UPLC characteristic spectrum Download PDFInfo
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- 238000004704 ultra performance liquid chromatography Methods 0.000 title claims abstract description 35
- 235000003956 Luffa Nutrition 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title claims abstract description 13
- 238000010276 construction Methods 0.000 title claims abstract description 11
- 238000001228 spectrum Methods 0.000 title claims description 24
- 244000050983 Luffa operculata Species 0.000 title 1
- 238000000605 extraction Methods 0.000 claims abstract description 43
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 241000219138 Luffa Species 0.000 claims abstract description 16
- 239000002904 solvent Substances 0.000 claims abstract description 16
- 239000012488 sample solution Substances 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 150000007524 organic acids Chemical class 0.000 claims abstract description 5
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims abstract description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 39
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 18
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 14
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 239000000706 filtrate Substances 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 claims description 7
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims description 7
- 235000004883 caffeic acid Nutrition 0.000 claims description 7
- 229940074360 caffeic acid Drugs 0.000 claims description 7
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- WKOLLVMJNQIZCI-UHFFFAOYSA-N vanillic acid Chemical compound COC1=CC(C(O)=O)=CC=C1O WKOLLVMJNQIZCI-UHFFFAOYSA-N 0.000 claims description 5
- TUUBOHWZSQXCSW-UHFFFAOYSA-N vanillic acid Natural products COC1=CC(O)=CC(C(O)=O)=C1 TUUBOHWZSQXCSW-UHFFFAOYSA-N 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 239000012085 test solution Substances 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 7
- 239000013558 reference substance Substances 0.000 abstract description 7
- 238000003908 quality control method Methods 0.000 abstract description 3
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 244000302544 Luffa aegyptiaca Species 0.000 description 21
- 235000009814 Luffa aegyptiaca Nutrition 0.000 description 20
- 239000000523 sample Substances 0.000 description 19
- 238000011835 investigation Methods 0.000 description 17
- 239000000463 material Substances 0.000 description 17
- 230000014759 maintenance of location Effects 0.000 description 12
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 10
- 239000003814 drug Substances 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000003643 water by type Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000004952 Polyamide Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920002647 polyamide Polymers 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 229920000057 Mannan Polymers 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- -1 galactans Polymers 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- LUEWUZLMQUOBSB-GFVSVBBRSA-N mannan Chemical class O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-GFVSVBBRSA-N 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 239000011265 semifinished product Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a construction and detection method of a luffa UPLC characteristic map, which comprises the following steps: (1) preparation of a sample solution: extracting retinervus Luffae fructus with extraction solvent to obtain sample solution; (2) establishing a characteristic map: sucking the sample solution, injecting into an ultra-high performance liquid chromatograph, and measuring to obtain a UPLC characteristic map; (3) assignment of chromatographic peaks: comparing with the reference substance, and assigning the common peak. The invention adopts the ultra-high performance liquid chromatography and reasonably controls the chromatographic conditions to determine 7 characteristic peaks in total, wherein 4 peaks are regarded as organic acid components, the specificity is good, the characteristic of the luffa can be comprehensively reacted, a novel analysis means can be provided for the inherent quality control of the luffa, the detection is comprehensive, rapid and simple, the analysis efficiency is high, the solvent consumption is low, and the environmental pollution is small.
Description
Technical Field
The invention relates to a construction and detection method of a characteristic spectrum, in particular to a construction and detection method of a luffa UPLC characteristic spectrum.
Background
The research at present finds that the loofah mainly contains xylan, cellulose, mannans, galactans, lignin and the like. The loofah sponge of different varieties and production places has high polysaccharide content. The retinervus Luffae fructus contains various retinervus Luffae fructus saponins, flavonoids and phenols, proteins and amino acids, oils and fats, inorganic elements and organic acids.
The loofah sponge is a common traditional Chinese medicine, but has less research data on the loofah sponge and lacks an overall quality control means. The fingerprint spectrum or the characteristic spectrum of the traditional Chinese medicine is a comprehensive and quantifiable identification means, can be used for evaluating the quality of traditional Chinese medicine materials, traditional Chinese medicine preparations and semi-finished products, is a means for controlling the overall quality of the traditional Chinese medicine, and can systematically, integrally and exclusively represent the internal characteristics of the traditional Chinese medicine.
In the prior art, the research on the characteristic spectrum or the fingerprint spectrum of the loofah sponge is less, the establishment of the high performance liquid chromatography fingerprint spectrum of the loofah sponge and the standard fingerprint spectrum and the application thereof are the only 1 patent of Liu Lijuan and the like (CN 201510732396.3) on the aspect of medicinal materials, the chromatographic analysis time of the method is as long as 80 minutes, the baseline fluctuation is larger, the separation degree of chromatographic peaks is poor, and the chemical components corresponding to the peaks are not clear. In the preparation process of the sample, a polyamide chromatographic column is adopted, and the preparation process is complex through water washing and 30% ethanol elution, so that the rapid and massive detection is not facilitated.
Disclosure of Invention
The invention aims to: the invention aims to provide a construction and detection method for a luffa UPLC characteristic map, which is comprehensive, quick and simple to detect.
The technical scheme is as follows: the construction method of the luffa UPLC characteristic map comprises the following steps:
(1) Preparation of test solution: extracting retinervus Luffae fructus with extraction solvent to obtain sample solution;
(2) Establishing a characteristic map: sucking the sample solution, injecting into an ultra-high performance liquid chromatograph, and measuring to obtain a UPLC characteristic map;
(3) Assignment of chromatographic peaks: the common peaks were assigned to the comparison, and 4 peaks were identified as organic acid components, namely protocatechuic acid, parahydroxybenzoic acid, vanillic acid and caffeic acid.
Further, the extraction solvent in the step (1) is methanol, water, and any one of 30-70% methanol aqueous solution or 30-70% ethanol aqueous solution by volume concentration.
Further, the extraction mode in the step (1) is ultrasonic treatment, shaking extraction or heating reflux, and the extraction time is 15-60min.
Further, the conditions of the ultra performance liquid chromatography in the step (2) are as follows: the chromatographic column takes octadecyl bonded silica gel as a filler; acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution or water is taken as a mobile phase B, and elution is carried out according to the following gradient: 0min-3min-9min-15min-21min-28min-30min, mobile phase A:2% -3% -6% -8% -9% -10%.
Further, the column temperature of the chromatographic column is 30-40 ℃; the detection wavelength is 235 nm-260 nm; the flow rate of the mobile phase is 0.25-0.35ml/min.
Further, the UPLC characteristic spectrum comprises 7 characteristic peaks, peak 4 is taken as a reference peak, and the relative retention time of peaks 1 to 7 is 0.43+/-10%, 0.69+/-10%, 0.90+/-10%, 1.00+/-10%, 1.08+/-10%, 1.58+/-10% and 1.82+/-10% respectively.
Further, the 4 peaks in step (3) correspond to peak 1, peak 2, peak 4 and peak 5, respectively, of the 7 characteristic peaks.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: (1) comprehensive, rapid and simple detection; the invention adopts ultra-high performance liquid chromatography, reasonably controls the chromatographic conditions, uses acetonitrile as a mobile phase A, uses 0.1% phosphoric acid solution or water as a mobile phase B for gradient elution, and can complete the whole analysis process within 30 minutes. The ethyl acetate is adopted for extraction and concentration in sample treatment, so that the elution is quicker and more convenient than the elution by adopting a polyamide chromatographic column, meanwhile, the interference of impurities on chromatographic peaks is reduced, and the response value of the chromatographic peaks is improved. 7 characteristic peaks are determined in total, wherein 4 peaks are identified as organic acid components, the specificity is good, the characteristic of the retinervus Luffae fructus can be comprehensively reflected, a new analysis means can be provided for the inherent quality control of the retinervus Luffae fructus, and the purposes of identifying retinervus Luffae fructus of different production places and different sources are achieved; (2) The method is simple, good in reproducibility, accurate, reliable and convenient to operate, and compared with HPLC, the method has the advantages of high analysis efficiency, less solvent consumption and less environmental pollution.
Drawings
FIG. 1 is a 3D UPLC chromatogram of retinervus Luffae fructus under full-wavelength scanning;
FIG. 2 is a UPLC chromatogram of retinervus Luffae fructus at 230-270 nm wavelength;
FIG. 3 shows UPLC chromatograms of retinervus Luffae fructus under different extraction solvents;
FIG. 4 shows UPLC chromatograms of retinervus Luffae fructus under different extraction modes;
FIG. 5 shows UPLC chromatograms of retinervus Luffae fructus under different extraction times;
FIG. 6 shows UPLC chromatograms of retinervus Luffae fructus under different extraction times;
FIG. 7 is a graph of the result of reference assignment;
fig. 8 is a UPLC superposition chart of retinervus Luffae fructus;
fig. 9 is a control chart of the luffa vegetable sponge material UPLC;
FIG. 10 shows UPLC chromatograms of retinervus Luffae fructus under different chromatographic column conditions;
FIG. 11 shows UPLC chromatograms of retinervus Luffae fructus under different column temperature conditions;
FIG. 12 is a UPLC chromatogram of retinervus Luffae fructus under different flow rates;
fig. 13 shows the UPLC chromatograms of retinervus Luffae fructus under different instrument conditions.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1
The construction method of the UPLC characteristic spectrum of the luffa medicinal material comprises the following steps:
1 instrument, reagent and sample
Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatograph system; waters Quaternary Solvent Manager quaternary pump; sample Manager-FTN autosampler; waters UPLC PDA detector; an Empower 3 chromatography workstation; agilent G6530 Accurate-Mass Q-TOF Mass spectrometer (Agilent company); agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent company); agilent Technologies 1290 Infinicity ultra-high performance liquid chromatograph; 1290DAD diode array detector; 1290MCT column incubator; 1290Vialsampler autosampler; 1290Fiexible pump quaternary pump; open lab CDS 2.3 chromatographic workstation. METTLER TOLEDO XP6 parts per million of the day; ME 204E-type electronic analytical balance; KQ-250B ultrasonic cleaner; a Milli-Q IQ pure water system; AS165W type centrifuge; GKC114 water-controlled bath. Acetonitrile; the water is ultrapure water; phosphoric acid; protocatechuic acid; vanillic acid; caffeic acid; p-hydroxybenzoic acid.
Determination of 2 chromatographic conditions
2.1 determination of detection wavelength
About 1.5g of the loofah sponge medicinal material is taken, precisely weighed, 25ml of water is added, the mixture is heated and refluxed for 60 minutes, cooled and filtered, the filtrate is transferred into a separating funnel, residues and containers are washed with a small amount of water for times, washing liquid is merged into the separating funnel, the washing liquid is extracted twice by shaking with ethyl acetate, 25ml of ethyl acetate liquid is combined each time, and the mixture is evaporated to dryness in a water bath. Dissolving the residue with 70% methanol water solution by volume, transferring to 5ml measuring flask, adding 70% methanol water solution by volume to scale, shaking, filtering, and collecting filtrate.
The sample solutions were subjected to gradient elution according to Table 1, and absorption spectra in the range of 200 to 400nm were recorded.
TABLE 1 gradient elution table
As shown in the figures 1 and 2, the chromatographic peaks detectable in the loofah sponge sample are more in the range of 230-270 nm, and the separation effect is better. Of these, 254nm may be the preferred detection wavelength.
2.2 preparation of sample solutions
(1) Investigation of the extraction solvent
About 1.5g of loofah sponge medicinal material powder is taken, 5 groups of loofah sponge medicinal material powder are taken and precisely weighed, placed in a conical flask with a plug, respectively and precisely added with 25ml of water, 30% by volume of methanol aqueous solution, 50% by volume of methanol aqueous solution, 70% by volume of methanol aqueous solution and methanol, heated and refluxed for 60 minutes, cooled, filtered, evaporated to dryness, dissolved with 25ml of water and transferred into a separating funnel, the residue and a container are washed with a small amount of water for times, the washing liquid is merged into the separating funnel, extracted twice by shaking with ethyl acetate, 25ml of ethyl acetate liquid each time is combined, and evaporated in a water bath. Dissolving the residue with 70% methanol water solution by volume, transferring to 5ml measuring flask, adding 70% methanol water solution by volume to scale, shaking, filtering, and collecting filtrate. The results are shown in FIG. 3.
Sample injection is carried out according to the chromatographic condition under the item of 2.1, and the investigation results of different extraction solvents of the loofah characteristic spectrum are shown in table 2 and fig. 3.
TABLE 2 loofah characteristic pattern extraction solvent investigation (peak area/sample size)
Experimental results: as can be seen from UPLC diagrams of different extraction solvents, other extraction solvents except high-concentration methanol are located in 7 chromatographic peaks, and the ratio of the total peak area of the 7 tentative chromatographic peaks to the sample weighing of the extraction solvent is calculated, so that the ratio of the total peak area to the sample weighing is relatively high during water extraction, and the ratio can be used as a preferable extraction solvent.
(2) Investigation of the extraction method
About 1.5g of loofah sponge medicinal material powder is taken, 3 groups are parallel, precisely weighed, placed in a conical flask with a plug, precisely added with 25ml of water, respectively subjected to ultrasonic treatment (power 250W, frequency 40 kHz), shaking extraction, heating reflux for 60 minutes, cooling, filtering, transferring filtrate into a separating funnel, washing residues and a container with a small amount of water for several times, merging washing liquid into the separating funnel, shaking extraction twice with ethyl acetate for 25ml each time, merging ethyl acetate liquid, and evaporating in a water bath to dryness. Dissolving the residue with 70% methanol water solution by volume, transferring to 5ml measuring flask, adding 70% methanol water solution by volume to scale, shaking, filtering, and collecting filtrate. Sample injection is carried out according to the chromatographic condition under the item of 2.1, and the investigation results of different extraction modes of the loofah characteristic spectrum are shown in figure 4.
Experimental results: as can be seen by comparing the chromatograms of different extraction modes, only the chromatographic peak of the heating reflux is more complete, so the heating reflux can be used as the preferred extraction mode of the characteristic map of the loofah sponge medicinal material.
(3) Investigation of extraction time
About 1.5g of loofah sponge medicinal material powder is taken, 4 groups are parallel, precisely weighed, placed in a conical flask with a plug, precisely added with 25ml of water, heated and refluxed for 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively, cooled, filtered, and the filtrate is transferred to a separating funnel, residues and containers are washed with a small amount of water, the washing liquid is merged into the separating funnel, extracted twice with 25ml of ethyl acetate by shaking, and the ethyl acetate liquid is combined and evaporated in a water bath. Dissolving the residue with 70% methanol water solution by volume, transferring to 5ml measuring flask, adding 70% methanol water solution by volume to scale, shaking, filtering, and collecting filtrate.
Sample injection is carried out according to the chromatographic condition under the item of 2.1, and the investigation results of different extraction times of the obtained luffa characteristic spectrum are shown in a table 3 and a figure 5.
TABLE 3 investigation of retinervus Luffae fructus characteristic spectrum extraction time (peak area/sample size)
Experimental results: from the results, it was found that the ratio of the total peak area of each chromatographic peak to the sample amount at different extraction times was not significantly different from the extraction efficiency at other times than 30 minutes, and 60 minutes was the preferred extraction time.
(4) Investigation of extraction time
About 1.5g of loofah sponge medicinal material powder is taken, 3 groups are parallel, precisely weighed, placed in a conical bottle with a plug, precisely added with 25ml of water, heated and refluxed for 60 minutes, cooled, filtered, transferred into a separating funnel, the residues and the container are washed with a small amount of water, the washing liquid is merged into the separating funnel, and extracted with ethyl acetate respectively for 1 time, 2 times and 3 times by shaking, 25ml of each time, combined with ethyl acetate liquid and evaporated to dryness in a water bath. Dissolving the residue with 70% methanol water solution by volume, transferring to 5ml measuring flask, adding 70% methanol water solution by volume to scale, shaking, filtering, and collecting filtrate. Sample injection is carried out according to the chromatographic condition under the item of 2.1, and the investigation results of different extraction times of the obtained loofah characteristic spectrum are shown in table 4 and fig. 6.
Table 4 comparison of the results of the peak patterns of the retinervus Luffae fructus medicinal material with different extraction times
Experimental results: from the results, the ratio of the total peak area of each chromatographic peak to the sample amount at different times of extraction was found to be smaller for two times of extraction than for three times of extraction, so that the number of times of extraction was 2 times as a preferable parameter.
3 building a characteristic spectrum
3.1 precisely sucking 2 μl of each batch of the sample solution, and injecting into a liquid chromatograph.
3.2, measuring 16 batches of luffa medicinal materials, analyzing and comparing to obtain a UPLC characteristic spectrum formed by common characteristic peaks, wherein the UPLC characteristic spectrum comprises 7 common characteristic peaks as shown in figure 7.
The selected multiple batches of samples are led into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.0 version) issued by the national formulary committee, a time window width is selected to be 0.1min, a contrast map is generated by median, mark peaks are matched after multipoint correction, a luffa medicinal material characteristic map sharing mode is generated, a peak 4 is taken as a reference peak, and the relative retention time and the relative peak area of a sharing peak of the characteristic map are calculated, wherein the table is shown in tables 5-6.
TABLE 5 relative Retention time of 16 batches of retinervus Luffae fructus
TABLE 6 relative peak area of 16 batches of retinervus Luffae fructus
The result shows that the retention time ratio of the characteristic peaks in the sample is stable, the RSD is less than 2%, the difference of the relative peak areas of the characteristic peaks is large, and the large difference in the relative amounts of the components of different batches of products is indicated, so that the relative peak areas of the characteristic peaks are temporarily not included in the standard.
4 assignment of common Peak
Taking appropriate amounts of protocatechuic acid reference substance, p-hydroxybenzoic acid reference substance, vanilloid reference substance and caffeic acid reference substance, and respectively adding methanol to obtain solutions containing protocatechuic acid 5 μg, p-hydroxybenzoic acid 10 μg, vanilloid 5 μg and caffeic acid 5 μg per 1 ml. 2 μl of each of the control solution and the sample solution was precisely sucked, and injected into the liquid chromatograph. The retention time and ultraviolet spectrum of peak 1, peak 2, peak 4, peak 5 in the sample are respectively matched with that of reference substance protocatechuic acid, parahydroxybenzoic acid, vanillic acid and caffeic acid reference substance solutions, so that they are respectively attributed to protocatechuic acid, parahydroxybenzoic acid, vanillic acid and caffeic acid. See in particular fig. 9.
Example 2
Methodology validation of feature map construction methods
(1) Precision investigation
The same sample solution in example 1 was continuously sampled 6 times, and the relative retention time and RSD value (table 7, table 8) of the relative peak area of 7 common characteristic peaks were calculated to be less than 3% by taking peak 4 as a reference peak, indicating that the instrument method was stable and the precision was good.
TABLE 7 precision experimental results (relative retention time)
Table 8 results of the precision experiments (relative peak area)
(2) Repeatability investigation
The same batch of luffa powder in example 1 is taken, 6 parts of luffa powder are precisely weighed, 6 parts of test solution are prepared in parallel according to the test preparation method in example 1, sample injection is carried out respectively, the peak number 4 is taken as a reference peak, and the relative retention time and the RSD value (in tables 9 and 10) of the relative peak areas of 7 common peaks are calculated to be less than 3%, which indicates that the repeatability of the construction method is good.
TABLE 9 repeatability test results (relative retention time)
TABLE 10 repeatability test results (relative peak area)
(3) Stability investigation
Taking the same sample solution, respectively carrying out sample injection analysis at 0, 4, 8, 12, 16, 20 and 24 hours, taking No. 4 peak as a reference peak, and calculating relative retention time and RSD values (table 11 and table 12) of relative peak areas of 7 common peaks to be less than 3%, wherein the sample solution is stable at least within 24 hours.
TABLE 11 stability test results (relative retention time)
TABLE 12 stability test results (relative peak area)
(4) Durability inspection
1) Investigation of different chromatographic columns
The 3 columns were compared, respectively: waters Acquity CORTECS UPLC T3 (100 mm×2.1mm,1.6 μm), agilent SB C18 RRHD (100 mm×2.1mm,1.8 μm), agilent Ecplise C18 (100 mm×2.1mm,1.8 μm), effect of 3 chromatographic columns on retinervus Luffae fructus medicinal material characteristic spectrum durability.
The result is shown in figure 10, and the experimental result shows that the separation effect of each peak of different types of chromatographic columns is better. Comparison of 3 columns revealed that the relative retention time of each peak was relatively stable.
2) Investigation of different temperatures
Comparing the different temperatures, respectively: the durability of the characteristic spectrum of the loofah sponge medicinal material is affected at 30 ℃,35 ℃ and 40 ℃.
The results are shown in FIG. 11, and the results show that the analysis method has better durability at the column temperature of 30-40 ℃. Smaller column temperature variations can meet system applicability requirements.
3) Investigation of different flow rates
Comparing the different flow rates, respectively: the influence of 0.25ml/min, 0.30ml/min and 0.35ml/min on the durability of the loofah sponge medicinal material map.
As a result, as shown in FIG. 12, the flow rate was in the range of 0.25 to 0.30ml/min, and small flow rate variation was able to meet the system applicability requirement. The durability is good.
4) Investigation by different instruments
The present study examined the separation effect of two different models of instruments (Waters UPLC and Agilent 1290).
As a result, as shown in FIG. 13, 7 tentative chromatographic peaks of the retinervus Luffae fructus can be separated well and the durability is good in different instruments.
Claims (4)
1. The construction method of the luffa UPLC characteristic map is characterized by comprising the following steps:
(1) Preparation of test solution: adding a luffa into an extraction solvent for extraction, wherein the extraction solvent is any one of methanol, water and 30-70% methanol aqueous solution or 30-70% ethanol aqueous solution, cooling, filtering, transferring the filtrate into a separating funnel, washing residues and a container with water for times, merging the washing liquid into the separating funnel, shaking and extracting twice with ethyl acetate, merging the ethyl acetate solutions, evaporating in a water bath, dissolving the residues by adding 70% methanol aqueous solution with volume concentration, transferring the solution into a measuring flask, adding 70% methanol aqueous solution with volume concentration to a scale, shaking uniformly, filtering, and taking the subsequent filtrate to obtain a sample solution;
(2) Establishing a characteristic map: sucking the sample solution, injecting into an ultra-high performance liquid chromatograph, and measuring to obtain a UPLC characteristic map; the conditions of the ultra-high performance liquid chromatography are as follows: the chromatographic column takes octadecyl bonded silica gel as a filler; acetonitrile is taken as a mobile phase A, 0.1% phosphoric acid solution or water is taken as a mobile phase B, and elution is carried out according to the following gradient: 0min-3min-9min-15min-21min-28min-30min, mobile phase A:2% -3% -6% -8% -9% -10%;
(3) Assignment of chromatographic peaks: the common peaks were assigned to the comparison, and 4 peaks were identified as organic acid components, namely protocatechuic acid, parahydroxybenzoic acid, vanillic acid and caffeic acid.
2. The method for constructing the UPLC characteristic spectrum of the luffa as claimed in claim 1, wherein the extraction mode in the step (1) is ultrasonic treatment, shaking extraction or heating reflux, and the extraction time is 15-60min.
3. The method for constructing a UPLC characteristic spectrum of retinervus Luffae fructus according to claim 1, wherein the column temperature of the chromatographic column is 30-40deg.C; the detection wavelength is 235 nm-260 nm.
4. The method for constructing a UPLC profile of retinervus Luffae fructus according to claim 1, wherein the flow rate of mobile phase is 0.25-0.35ml/min.
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| CN102426211A (en) * | 2011-07-20 | 2012-04-25 | 石河子大学 | Method for quantitatively determining phenolic acid compounds in barley grains |
| CN105203670A (en) * | 2015-11-02 | 2015-12-30 | 黑龙江大学 | Establishing method of HPLC (high performance liquid chromatography) fingerprints of loofah sponge, standard fingerprints established with method and application |
| CN112730671A (en) * | 2020-12-23 | 2021-04-30 | 江阴天江药业有限公司 | Ultra-high performance liquid chromatography detection method for loofah sponge standard decoction and application of ultra-high performance liquid chromatography detection method |
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| CN102426211A (en) * | 2011-07-20 | 2012-04-25 | 石河子大学 | Method for quantitatively determining phenolic acid compounds in barley grains |
| CN105203670A (en) * | 2015-11-02 | 2015-12-30 | 黑龙江大学 | Establishing method of HPLC (high performance liquid chromatography) fingerprints of loofah sponge, standard fingerprints established with method and application |
| CN112730671A (en) * | 2020-12-23 | 2021-04-30 | 江阴天江药业有限公司 | Ultra-high performance liquid chromatography detection method for loofah sponge standard decoction and application of ultra-high performance liquid chromatography detection method |
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